In vivo gene repair of point and frameshift mutations directed by chimericRNA/DNA oligonucleotides and modified single-stranded oligonucleotides

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chime ric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machine ry, the CO directs nucleotide exchange as episomal and chromosomal DNA. Sys tematic dissection of the CO revealed that the region of contiguous DNA bas es was the active component in the repair process, especially when the sing le-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome contai ning a mutated fusion gene encoding hygromycin resistance and eGFP expressi on was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified si ngle-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleoti de on the non-transcribed (non-template) strand displayed a 5-10-fold highe r level of activity. Expanding the length of the oligo-vector from 25 to 10 0 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.